Lotus japonicus SUNERGOS1 encodes a predicted subunit A of a DNA topoisomerase VI that is required for nodule differentiation and accommodation of rhizobial infection

A symbiotic mutant of Lotus japonicus, called sunergos1‐1 (suner1‐1), originated from a har1‐1 suppressor screen. suner1‐1 supports epidermal infection by Mesorhizobium loti and initiates cell divisions for organogenesis of nodule primordia. However, these processes appear to be temporarily stalled early during symbiotic interaction, leading to a low nodule number phenotype. This defect is ephemeral and near wild‐type nodule numbers are reached by suner1‐1 at a later point after infection. Using an approach that combined map‐based cloning and next‐generation sequencing we have identified the causative mutation and show that the suner1‐1 phenotype is determined by a weak recessive allele, with the corresponding wild‐type SUNER1 locus encoding a predicted subunit A of a DNA topoisomerase VI. Our data suggest that at least one function of SUNER1 during symbiosis is to participate in endoreduplication, which is an essential step during normal differentiation of functional, nitrogen‐fixing nodules.     

Lotus SHAGGY‐like kinase 1 is required to suppress nodulation in Lotus japonicus

Glycogen synthase kinase/SHAGGY‐like kinases (SKs) are a highly conserved family of signaling proteins that participate in many developmental, cell‐differentiation, and metabolic signaling pathways in plants and animals. Here, we investigate the involvement of SKs in legume nodulation, a process requiring the integration of multiple signaling pathways. We describe a group of SKs in the model legume Lotus japonicus (LSKs), two of which respond to inoculation with the symbiotic nitrogen‐fixing bacterium Mesorhizobium loti. RNAi knock‐down plants and an insertion mutant for one of these genes, LSK1, display increased nodulation. Ηairy‐root lines overexpressing LSK1 form only marginally fewer mature nodules compared with controls. The expression levels of genes involved in the autoregulation of nodulation (AON) mechanism are affected in LSK1 knock‐down plants at low nitrate levels, both at early and late stages of nodulation. At higher levels of nitrate, these same plants show the opposite expression pattern of AON‐related genes and lose the hypernodulation phenotype. Our findings reveal an additional role for the versatile SK gene family in integrating the signaling pathways governing legume nodulation, and pave the way for further study of their functions in legumes.     

Negative regulation of CCaMK is essential for symbiotic infection

One of the earliest responses of legumes to symbiotic signalling is oscillation of the calcium concentration in the nucleoplasm of root epidermal cells. Integration and decoding of the calcium‐spiking signal involve a calcium‐ and calmodulin‐dependent protein kinase (CCaMK) and its phosphorylation substrates, such as CYCLOPS. Here we describe the Lotus japonicus ccamk‐14 mutant that originated from a har1‐1 suppressor screen. The ccamk‐14 mutation causes a serine to asparagine substitution at position 337 located within the calmodulin binding site, which we determined to be an in vitro phosphorylation site in CCaMK. We show that ccamk‐14 exerts cell‐specific effects on symbiosis. The mutant is characterized by an increased frequency of epidermal infections and significantly compromised cortical infections by Mesorhizobium loti and also the arbuscular mycorrhiza fungus Rhizophagus irregularis. The S337 residue is conserved across angiosperm CCaMKs, and testing discrete substitutions at this site showed that it participates in a negative regulation of CCaMK activity, which is required for the cell‐type‐specific integration of symbiotic signalling.     

Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development

Arbuscular mycorrhiza (AM) fungi form nutrient‐acquiring symbioses with the majority of higher plants. Nutrient exchange occurs via arbuscules, highly branched hyphal structures that are formed within root cortical cells. With a view to identifying host genes involved in AM development, we isolated Lotus japonicus AM‐defective mutants via a microscopic screen of an ethyl methanesulfonate‐mutagenized population. A standardized mapping procedure was developed that facilitated positioning of the defective loci on the genetic map of L. japonicus, and, in five cases, allowed identification of mutants of known symbiotic genes. Two additional mutants representing independent loci did not form mature arbuscules during symbiosis with two divergent AM fungal species, but exhibited signs of premature arbuscule arrest or senescence. Marker gene expression patterns indicated that the two mutants are affected in distinct steps of arbuscule development. Both mutants formed wild‐type‐like root nodules upon inoculation with Mesorhizobium loti, indicating that the mutated loci are essential during AM but not during root nodule symbiosis.     

LjMOT1, a high‐affinity molybdate transporter from Lotus japonicus,is essential for molybdate uptake,but not for the delivery to nodules

Molybdenum (Mo) is an essential nutrient for plants, and is required for nitrogenase activity of legumes. However, the pathways of Mo uptake from soils and then delivery to the nodules have not been characterized in legumes. In this study, we characterized a high‐affinity Mo transporter (LjMOT1) from Lotus japonicus. Mo concentrations in an ethyl methanesulfonate–mutagenized line (ljmot1) decreased by 70–95% compared with wild‐type (WT). By comparing the DNA sequences of four AtMOT1 homologs between mutant and WT lines, one point mutation was found in LjMOT1, which altered Trp²⁹² to a stop codon; no mutation was found in the other homologous genes. The phenotype of Mo concentrations in F₂ progeny from ljmot1 and WT crosses were associated with genotypes of LjMOT1. Introduction of endogenous LjMOT1 to ljmot1 restored Mo accumulation to approximately 60–70% of the WT. Yeast expressing LjMOT1 exhibited high Mo uptake activity, and the K_m was 182 nm . LjMOT1 was expressed mainly in roots, and its expression was not affected by Mo supply or rhizobium inoculation. Although Mo accumulation in the nodules of ljmot1 was significantly lower than that of WT, it was still high enough for normal nodulation and nitrogenase activity, even for cotyledons‐removed ljmot1 plants grown under low Mo conditions, in this case the plant growth was significantly inhibited by Mo deficiency. Our results suggest that LjMOT1 is an essential Mo transporter in L. japonicus for Mo uptake from the soil and growth, but is not for Mo delivery to the nodules.     

Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 is essential for nodule,nectary, leaf and flower development

The NOOT‐BOP‐COCH‐LIKE (NBCL) genes are orthologs of Arabidopsis thaliana BLADE‐ON‐PETIOLE1/2. The NBCLs are developmental regulators essential for plant shaping, mainly through the regulation of organ boundaries, the promotion of lateral organ differentiation and the acquisition of organ identity. In addition to their roles in leaf, stipule and flower development, NBCLs are required for maintaining the identity of indeterminate nitrogen‐fixing nodules with persistent meristems in legumes. In legumes forming determinate nodules, without persistent meristem, the roles of NBCL genes are not known. We thus investigated the role of Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 (LjNBCL1) in determinate nodule identity and studied its functions in aerial organ development using LORE1 insertional mutants and RNA interference‐mediated silencing approaches. In Lotus, LjNBCL1 is involved in leaf patterning and participates in the regulation of axillary outgrowth. Wild‐type Lotus leaves are composed of five leaflets and possess a pair of nectaries at the leaf axil. Legumes such as pea and Medicago have a pair of stipules, rather than nectaries, at the base of their leaves. In Ljnbcl1, nectary development is abolished, demonstrating that nectaries and stipules share a common evolutionary origin. In addition, ectopic roots arising from nodule vascular meristems and reorganization of the nodule vascular bundle vessels were observed on Ljnbcl1 nodules. This demonstrates that NBCL functions are conserved in both indeterminate and determinate nodules through the maintenance of nodule vascular bundle identity. In contrast to its role in floral patterning described in other plants, LjNBCL1 appears essential for the development of both secondary inflorescence meristem and floral meristem.     

Auxin distribution in Lotus japonicus during root nodule development

For this work, Lotus japonicus transgenic plants were constructed expressing a fusion reporter gene consisting of the genes beta-glucuronidase (gus) and green fluorescent protein (gfp) under control of the soybean auxin-responsive promoter GH3. These plants expressed GUS and GFP in the vascular bundle of shoots, roots and leafs. Root sections showed that in mature parts of the roots GUS is mainly expressed in phloem and vascular parenchyma of the vascular cylinder. By detecting GUS activity, we describe the auxin distribution pattern in the root of the determinate nodulating legume L. japonicus during the development of nodulation and also after inoculation with purified Nod factors, N-naphthylphthalamic acid (NPA) and indoleacetic acid (IAA). Differently than white clover, which forms indeterminate nodules, L. japonicus presented a strong GUS activity at the dividing outer cortical cells during the first nodule cell divisions. This suggests different auxin distribution pattern between the determinate and indeterminate nodulating legumes that may be responsible of the differences in nodule development between these groups. By measuring of the GFP fluorescence expressed 21 days after treatment with Nod factors or bacteria we were able to quantify the differences in GH3 expression levels in single living roots. In order to correlate these data with auxin transport capacity we measured the auxin transport levels by a previously described radioactive method. At 48 h after inoculation with Nod factors, auxin transport showed to be increased in the middle root segment. The results obtained indicate that L. japonicus transformed lines expressing the GFP and GUS reporters under the control of the GH3 promoter are suitable for the study of auxin distribution in this legume.     

N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

Studies of protein N‐glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N‐glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N‐glycan patterns as documented using mass spectrometry and glycan‐recognising antibodies, indicating successful identification of null mutations in the target glyco‐genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3‐fucosyltransferase (Lj3fuct) mutant completely lacked α1,3‐core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N‐glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N‐acetylglucosaminyltransferase I, and α1,3‐fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N‐glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N‐glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian‐like N‐glycosylation features.     

Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

The nitrogen‐fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix^– mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix^– mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix^– mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (LjAPN1), encodes a nepenthesin‐type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain‐specific Fix^– phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen‐fixing) symbiosis in a rhizobial strain‐dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.     

Functional analysis of chimeric lysin motif domain receptors mediating Nod factor‐induced defense signaling in Arabidopsis thaliana and chitin‐induced nodulation signaling in Lotus japonicus

The expression of chimeric receptors in plants is a way to activate specific signaling pathways by corresponding signal molecules. Defense signaling induced by chitin from pathogens and nodulation signaling of legumes induced by rhizobial Nod factors (NFs) depend on receptors with extracellular lysin motif (LysM) domains. Here, we constructed chimeras by replacing the ectodomain of chitin elicitor receptor kinase 1 (AtCERK1) of Arabidopsis thaliana with ectodomains of NF receptors of Lotus japonicus (LjNFR1 and LjNFR5). The hybrid constructs, named LjNFR1–AtCERK1 and LjNFR5–AtCERK1, were expressed in cerk1‐2, an A. thaliana CERK1 mutant lacking chitin‐induced defense signaling. When treated with NFs from Rhizobium sp. NGR234, cerk1‐2 expressing both chimeras accumulated reactive oxygen species, expressed chitin‐responsive defense genes and showed increased resistance to Fusarium oxysporum. In contrast, expression of a single chimera showed no effects. Likewise, the ectodomains of LjNFR1 and LjNFR5 were replaced by those of OsCERK1 (Oryza sativa chitin elicitor receptor kinase 1) and OsCEBiP (O. sativa chitin elicitor‐binding protein), respectively. The chimeras, named OsCERK1–LjNFR1 and OsCEBiP–LjNFR5, were expressed in L. japonicus NF receptor mutants (nfr1‐1; nfr5‐2) carrying a GUS (β‐glucuronidase) gene under the control of the NIN (nodule inception) promoter. Upon chitin treatment, GUS activation reflecting nodulation signaling was observed in the roots of NF receptor mutants expressing both chimeras, whereas a single construct was not sufficient for activation. Hence, replacement of ectodomains in LysM domain receptors provides a way to specifically trigger NF‐induced defense signaling in non‐legumes and chitin‐induced nodulation signaling in legumes.     

The Lotus japonicus ndxgene family is involved in nodule function and maintenance

To elucidate the function of the ndx homeobox genes during the Rhizobium-legume symbiosis, two Lotus japonicus ndxgenes were expressed in the antisense orientation under the control of the nodule-expressed promoter Psenod12 in transgenic Lotus japonicus plants. Many of the transformants obtained segregated into plants that failed to sustain proper development and maintenance of root nodules concomitant with down-regulation of the two ndx genes. The root nodules were actively fixing nitrogen 3 weeks after inoculation, but the plants exhibited a stunted growth phenotype. The nodules on such antisense plants had under-developed vasculature and lenticels when grown on medium lacking nitrogen sources. These nodules furthermore entered senescence earlier than the wild-type nodules. Normal plant growth was resumed upon external addition of nitrogen. This suggests that assimilated nitrogen is not properly supplied to the plants in which the two ndx genes are down-regulated. The results presented here, indicate that the ndx genes play a role in the development of structural nodule features, required for proper gas diffusion into the nodule and/or transport of the assimilated nitrogen to the plant.     

The evolutionary appearance of non‐cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous β–glucosidases resulting from a crucial amino acid substitution

Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic α–hydroxynitrile glucosides lotaustralin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific β–glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non‐cyanogenic γ‐ and β–hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only β–glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site‐directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non‐flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L. japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic β–glucosidase, resembling BGD2, and required no more than a single amino acid substitution.     

Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus

Use of insertion mutants facilitates functional analysis of genes, but it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics in most plant species. The main challenge is developing efficient high-throughput procedures for both mutagenesis and identification of insertion sites. To date, only floral-dip T-DNA transformation of Arabidopsis has produced independent germinal insertions, thereby allowing generation of mutant populations from seeds of single plants. In addition, advances in insertion detection have been hampered by a lack of protocols, including software for automated data analysis, that take full advantage of high-throughput next-generation sequencing. We have addressed these challenges by developing the FSTpoolit protocol and software package, and here we demonstrate its efficacy by detecting 8935 LORE1 insertions in 3744 Lotus japonicus plants. The identified insertions show that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to homogenous gene targeting and exonic insertion preference. As LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating progeny generates a complete mutant population. This ease of LORE1 mutagenesis, combined with the efficient FSTpoolit protocol, which exploits 2D pooling, Illumina sequencing and automated data analysis, allows highly cost-efficient development of a comprehensive reverse genetic resource.     

Grafting analysis indicates that malfunction of TRICOT in the root causes a nodulation-deficient phenotype in Lotus japonicus

Leguminous plants develop root nodules in symbiosis with soil rhizobia. Nodule formation occurs following rhizobial infection of the host root that induces dedifferentiation of some cortical cells and the initiation of a new developmental program to form nodule primordia. In a recent study, we identified a novel gene, TRICOT (TCO), that acts as a positive regulator of nodulation in Lotus japonicus. In addition to its role in nodulation, tco mutant plants display pleiotropic defects including abnormal shoot apical meristem formation. Here, we investigated the effect of the tco mutation on nodulation using a grafting approach. The results strongly indicate that the nodulation-deficient phenotype of the mutant results from malfunction of the TCO gene in the root.